HIV infection is a global pandemic, the extent of which is
difficult to evaluate. HIV infection at the present includes all the
inhabited continents. It has become a huge problem for the World Heath
Organization (WHO), overtaking cancers and cardiovascular diseases as a
cause of morbidity. In the Russian Federation there were 461,756
HIV-infected patients officially registered on December 31, 2008. In the
Smolensk region (the population on January 1, 2007 was 1,059,000) there
were approximately 1,226 and specialists are confident that this was
only the 'tip of the iceberg' (Doronin, Makeyenkov, & Yu,
2007).

The high-risk groups for the transmission of HIV are people in
their 20th and 30th decades. This prevalence is because of a high rate
of unprotected sexual activity, together with intravenous drug use (Royce, Sena, Cates, & Cohen, 1997). Moreover, lack of knowledge
about safe sex techniques and the use of nonsterile syringes in drug use
help spread the virus significantly.

Severe immunodeficiency in humans with HIV infections makes them
prone to numerous opportunistic infections of different systems. The
most common targets of these infections are the respiratory tract, the
gastrointestinal tract, the central nervous system, the genitourinary
tract, and the skin. Recent data has shown that mortality in
HIV-infected patients is not only from HIV-related causes but also from
other causes, such as neoplasia, and liver and heart disease. These
increase with immunological impairment (Weber et al., 2005).
Opportunistic infections have gradually decreased in recent years in
HIV-infected patients, thanks to highly active antiretroviral therapy
(HAART), particularly in early diagnosis and treatment regimens.
However, in certain developing countries, HAART is not available to the
general population, especially in prisons, so opportunistic infections
are frequently encountered in these settings. Tuberculosis plays a
significant role as one of the commonly found infectious pathologies,
and in severely immunocompromised patients, it is typically found in a
destructive phase. Moreover, the respiratory tract, being the most
vulnerable for such infections, is often affected the most. Therefore,
bacterial pneumonia is a frequently found pathology and is often
exacerbated by viral, fungal, and protozoal superinfections (Furrer and
Fux, 2002). Our experience for the past six years has shown that
opportunistic infections are the most widespread in HIV-infected
patients who undergo delayed HAART or who do not receive any treatment.
In recent years, we have also found cases with neoplasia.

The aim of this study was to analyze the cases, and reveal the most
frequently occurring opportunistic diseases and their clinical and
morphological characteristics in patients who died following HIV
infection in the Smolensk region.

Materials and Methods

We used case histories and autopsy research protocols of 32
patients (27 males and five females) aged between 24 and 49 years (mean
31.1 years) who died following HIV infection in various hospitals in the
Smolensk region between 2003 and 2008. Autopsy specimens of various
organs were studied histologically and microbiologically.

Histopathology

Large tissue sections (1.0 x 0.5 x 0.5 cm) were taken during
autopsy, fixed in 10--15% formalin and embedded in paraffin wax for
sectioning. The standard protocol is shown in Table 1.

Microbiology

Microbiological study included cultures of blood, spleen and lungs
in cases of suspected sepsis. Bacterioscopy, histopathology of freshly
prepared cytology smears and virological investigations were used
routinely in diagnosing the infectious agents.

Histology

The standard staining protocol is shown in Table 2.

Results and Discussion

Staging of disease was performed following the guidelines of the
Russian classification of HIV and AIDS according to Pokrovskii et al.
(2001), which can be summarized as: stage 1, incubation period; stage 2,
primary manifestations; stage 3, latent period; stage 4, period of
secondary disease. Stage 4 is further divided into: substage 4A,
prevalence of superficial infections; substage 4B, prevalence of
visceral disease; substage 4C, generalized pathologies; and stage 5,
terminal. Accordingly, 10 patients (31%) were diagnosed as at stage 4B,
while 22 patients (69%) were diagnosed as stage 4C. The mode of
infection was revealed in 18 patients (56%): parenteral (12 patients)
and sexual (six patients), while in 14 patients, the mode of infection
could not be determined. Sixteen patients (50%) were diagnosed with HIV
infection in prison. Most (69%) patients had chronic hepatitis C. All
the patients were diagnosed with HIV infection before death, based on
HIV-specific enzyme-linked immunosorbent assay (ELISA) and
immunoblotting, and no patient survived for more than five years from
the time of detecting HIV seropositivity.

Pathology revealed that the cause of death was an infectious
process in all cases, and a neoplastic growth was detected in three
patients. The most frequently occurring during autopsy was tuberculosis:
miliary tuberculosis with affection of various internal organs (72% of
cases) and similarly progressive secondary tuberculosis of the lungs
(16%). In 9% of cases, miliary tuberculosis was accompanied by affected
meninges and 16% of patients had peritoneal infections. In all the
patients with tuberculosis, caseous changes had affected many groups of
visceral lymph nodes. Six patients suffered simultaneously from
pulmonary tuberculosis and pneumonia of bacterial etiology
(Staphylococcus, Pneumococcus and Klebsiella spp.). In 6% of cases, the
cause of death was adenogenous sepsis of bacterial etiology as a result
of cervical lymphadenitis and 12.5% patients died from abscess-forming
pneumonia. Three patients died from AIDS-related neoplasms. It is
important to note that a patient can suffer from multiple pathologies.
The opportunistic diseases revealed in our study are summarized in Table
3.

In 12 patients (37.5%), tuberculosis and pneumonia were diagnosed
during life (bacteriologically and radiologically). In the remaining 20
cases, the pathologies were revealed during postmortem investigation.

HIV infection is characterized by generalized lymphadenopathy,
which in its growth, passes through a sequence: hyperplasia, involution,
depletion, and sclerosis (Libman, 1987). Lymphadenopathy is one of the
earliest manifestations of HIV infection. In the stage of hyperplasia,
the lymph nodes were characterized by disorderly arranged multiple
follicles resembling a 'starry sky' picture caused by many
macrophages. Formation of multinuclear cells resembling symplasts merged
from lymphocytes affected by virus were rarely found. With the
progression of disease, lymphoid depletion became extensive and a
fibrovascular carcass was more evident. There was increasing vascularity
(angiomatosis) and macrophages in the pulp and sinuses.

As the damage to the immune status of an organism continues
following HIV infection, secondary diseases begin to appear and
manifested forms of infections and tumors become evident. One of the
most frequently revealed causes of death in these patients with HIV
infection was tuberculosis, with a prevalence of its generalized form,
extensive dissemination and acute progression of specific processes.
Tuberculosis is the most virulent infection that is manifested in such
cases, usually before other opportunistic infections, and HIV-infected
patients comprise one of the high-risk groups for infection.

Morphology of Tuberculosis in HIV-Infected Patients

There was a high prevalence of pulmonary and extrapulmonary
tuberculosis. The main forms of tuberculosis were generalized (44%),
disseminated (6%), fibro-cavernous (12.5%), caseous pneumonia (9%), and
others (tuberculosis of the central nervous system, cirrhotic
tuberculosis of lungs, and tuberculosis of the intestine, 4%). All forms
of tuberculosis seen in the terminal stages were actively progressive.
Various organs were affected, most often the lymph nodes, lungs, liver,
kidneys, spleen, intestine, and central nervous system. Tissue reactions
in the terminal stage were typical tuberculosis granulomas in only 20%
of cases, and in the remaining 80%, there were many foci of nonreactive
caseous necrosis.

Pulmonary tuberculosis

Pulmonary tuberculosis was manifested as a bilateral disseminated
type or polycavernous variant. In disseminated tuberculosis, foci of
specific lesions (granulomas) comprised large central zones of caseous
necrosis surrounded by a few inflammatory cells. Giant cells were rarely
found. Ziehl--Neelsen staining showed numerous acid-fast bacteria in the
foci of caseous necrosis. Thus, tuberculosis was in the progressive
phase and highly active.

Macroscopic study of the lungs often revealed miliary bilateral
disseminated tuberculosis, but macronodular dissemination and caseous
pneumonia were rare. Dissemination occurred in most of the cases of
bilateral, with a predominance of micronodular, miliary and submiliary
types, although large foci (~1 cm diameter and
mixed--macro--micronodular--dissemination) was common (Fig. 1). We found
tuberculous foci in all parts of the lungs, evenly spread to the whole
organ or localized to one of the lobes. The intrathoracic lymph nodes
were also affected, enlarged (3--4 cm in diameter), and aggregated; on
sectioning, they were partially or totally replaced by caseous masses.

[FIGURE 1 OMITTED]

The characteristic feature was a predominance of alterative and
exudative changes with the absence of a productive component of
inflammation or its minimal manifestation (Fig. 2). The latter is marked
by the absence of signs of encapsulation and organization of
inflammatory foci. Typically, there were few multinuclear giant cells of
Langerhans. Granulomas were infrequent and there was little evidence of
the wave-like course of disease characterizing the classical variant of
tuberculosis. The specific foci of inflammation had monomorphic structures and consisted of a few Langerhans cells surrounded by
infiltrates of macrophages and lymphocytes (Fig. 3).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Initially, there was formation of colonies of Mycobacteria in the
pulmonary parenchyma, which was accompanied by cellular infiltration
with a significant predominance of polymorphonuclear leucocytes. The
cells had phagocytosed the bacteria and this step was marked by
karyorrhexis. Later, this process was characterized by massive breakdown
of leucocytes and formation of necrosis and microabscesses. Tissue
sections stained by Ziehl--Neelsen showed numerous acid-fast bacteria in
the foci of caseous necrosis (Fig. 4A--B).

[FIGURE 4 OMITTED]

An extensive exudative reaction in the form of serous-fibrinous
pneumonia or fibrinous-purulent pneumonia with predominance of
neutrophilic leucocytes was detected at the peripheries of caseous foci.
In some fields of view, there were minute foci of accumulations of foamy
macrophages that are characteristic for typical tuberculous
inflammation.

There was an increase in the thickness of the pleura caused by
extensive hyperemia and edema. Cell infiltrates differed according to
localization and intensity: perivascular, diffused, diffused-focal and
with various stages of intensity. Cells present in nonspecific
infiltrates were leucocytes, macrophages and small numbers of
lymphocytes or specific granulomatous foci (Fig. 3).

In mediastinal lymph nodes, in many cases there was partial or
total caseous lymphadenitis with the spread of inflammatory processes to
the surrounding soft tissues. There was fusion of purulent and necrotic
masses and absence of productive and granulomatous reactions in the
foci. Evident reduction of follicular structures and lymphoid depletion
was a characteristic feature of these lymph nodes. In most cases,
macroscopic detection of tuberculous changes in lungs was extremely
difficult, but histopathology revealed miliary and submiliary necrotic
foci. Typically, these showed alterations with the absence of typical
granulomas, monomorphic-type foci and effects on blood vessels in the
form of vasculitis. Moreover, morphological changes in lungs were
characterized by an acute progression of the disease with absence of a
wave-like course of pathology, absence of typical granulomatous tissue
reactions, and specificity of inflammatory changes.

Extrapulmonary tuberculosis

Tuberculous meningitis was found in three cases, grossly
characterized by typical basilar localization with insignificant
gray-white exudates and tubercles in the subarachnoid space. Microscopic
examination of the meninges revealed evident hyperemia and edema
accompanied by alterative reactions. The latter was manifested as areas
of coagulative/caseous necrosis extensively infiltrated by polymorphs,
lymphocytes, and macrophages. The endothelium of blood vessels was
edematous with pale hypertrophied nuclei and signs of desquamation of
endothelial cells in the vascular lumen. Various types of vasculitis
such as endovasculitis, panvasculitis, thrombovasculitis, and
perivasculitis were evident. Perivasculitis was more often present with
edema and excessive mononuclear, neutrophilic, eosinophilic, and plasma
cell infiltrates in all layers of the vessel wall (Fig. 5).

Five patients (16%) had tuberculous peritonitis. Unless this
localization was included as a component of generalized tuberculosis,
specific peritonitis was recorded as the leading cause of death. It is
important to note that none of these cases was diagnosed before death,
as the clinical picture was confused with chronic pain syndrome,
intestinal obstruction, or recurrent intestinal infection. The source of
dissemination was assumed hematogenous from the lungs in three cases,
lymphogenous from affected mesenteric lymph nodes in one case, and from
microperforations of multiple tuberculous ulcers in one patient. At
autopsy, peritonitis had an adhesive character and in some cases it had
resulted in the formation of fibrous tissue between intestinal loops.
Despite the longer course of inflammatory processes in the peritoneum,
microscopically there was a typical alterative character with miliary
foci and extensive areas of caseous necrosis without expressed
productive reaction. Moreover, granulomas were found in the
subadventitia of the large intestine in two cases of generalized
tuberculosis. However, there were no signs of peritonitis in these two
cases on visual inspection. We suppose that in these cases, the process
was present at an initial stage, but the death of the patients from
other causes interrupted the course of tuberculous peritonitis.

[FIGURE 5 OMITTED]

N, male, aged 25 years.) H & E x 240.

Extrapulmonary foci of tuberculous infection were detected in 23
patients (44%) as a component of a generalized type of tuberculosis.
Monomorphic miliary foci of caseous necrosis were found in various
internal organs, more often in the spleen, kidneys, and liver in
descending frequency, and rarely in the meninges, peritoneum, ovaries,
pancreas, or adrenal glands. As a whole, in cases of generalized
tuberculosis (14 patients), Mycobacteria caused alterative and exudative
reactions simultaneously in several organs (the mean number of organs
involved with Mycobacterium was 5.4). All the foci were suspected to be
spread hematogenously from lungs. Histopathology of the organs revealed
miliary nodules of caseous necrosis with rare giant cells, as in other
organs. In many cases, signs of affective reactions were not visible by
visual inspection. In the spleen, the foci of caseous necrosis had a
tendency to fuse and often covered up to 50% of the cut surface.

Bacterial Pneumonia

Bacterial pneumonia was the second most common cause of death of
these patients (25%). There was a broad spectrum of causative agents of
pneumonia revealed by microbiology. Besides typical microflora,
bacterial pneumonia can be caused by opportunistic agents, which are
activated in conditions of general immunodeficiency and extensive
decrease in local resistance of respiratory tract and various agents
simultaneously. Figure 6A--B shows typical histopathology.

Often, the course of pneumonia in these HIV-infected patients had a
tendency to form microabscesses, and in such cases, the microscopic
changes resembled microfocal dissemination in pulmonary tuberculosis. In
microabscesses, purulent necrotic foci were found with expressed
perifocal exudative reaction, which strengthened their resemblance to
pulmonary tuberculosis in HIV-infected patients. The causative agents of
pneumonia were Klebsiella pneumoniae, Staphylococcus aureus,
Streptococcus pneumoniae and E. coli.

H & E and Gram staining of sections of lungs helped in
revealing nonspecific microflora (Fig. 7). At autopsy, staining of
smears of lung sections using Romanovskii--Giemsa and by Gram staining
also helps in establishing the nonspecific character of microflora in
cases of bacterial pneumonia, because at autopsy, Ziehl--Neelsen
staining of smears does not reveal causative agents of tuberculosis.
Microbiology (bacteriological culture of lung tissue) helps in revealing
the nature of the causative agents of pneumonia most accurately.

[FIGURE 7 OMITTED]

Pneumocystis carinii Pneumonia

Pneumocystis carinii typically produces pneumonia that is
widespread throughout the lungs with a chronic course of disease and
rapid progression. Pulmonary pneumocystosis is a disease caused by
intense multiplication of relatively pathogenic single-celled saprophyte Pneumocystis carinii in the human respiratory tract (Garcia, 1993). The
terminal period of pneumocystosis is pneumonia, manifested in the later
stages of HIV infection, and leads to death in most cases. The gross
appearance resembled pneumonic consolidation. The cut surface of the
lung was pale pink with scattered areas of congestion and, rarely,
hemorrhages.

Microscopically, in the edematous stage, a characteristic,
homogenous, foamy protein containing eosinophilic exudates in cysts with
fistulae was found in the alveolar lumen. This is a pathognomonic sign
of pneumocystic pneumonia (Fig. 8). Neutrophils, macrophages, and plasma
cells were detected around the collections of Pneumocystis carinii. In
some fields of view, pink foamy alveolar exudates were present and the
interstitial inflammatory changes were minimal.

[FIGURE 8 OMITTED]

Cytomegaloviral Infections

CMV infection is caused by a DNA virus of the herpes virus group.
Infection proceeds diversely from latent infection to severe acute
generalization in the later stages of HIV infection (Wang, Huong, Chiu,
Raab-Traub, & Huang, 2003). Microscopically, CMV infections appear
as characteristic metamorphosis of alveolar and bronchial epithelium.
They are usually well determined and do not cause difficulties in
diagnosis. The persistence of viruses in the epithelial cells leads to
cytomegalic giant cell metamorphosis.

Epithelial cells increased in size up to 25--40 [micro]m. About
1--2 nuclear inclusions were detected containing viral particles in the
chromatin in each cell and there was a thin perinuclear clear halo. The
nucleus of each affected cell was usually eccentrically positioned and
the cell border was not prominent. Additionally, the cytoplasm of
affected cells contained coarse dark basophilic bodies.

[FIGURE 9 OMITTED]

[FIGURE 10 OMITTED]

Characteristic infiltrative changes and cytomegalic transformations
were numerous. We found focal accumulations of serous fluid and protein
masses in the alveolar cavities with admixtures of macrophages and
erythrocytes, moderate cytomegalic transformation of alveolar and
bronchial epithelial cells (2--3 typical cells in the form of an
'owl-eye' in the field of view), and weak infiltrations of
interstitial tissue (Fig. 9).

If the lung changes consisted of diffuse persisting alveolitis with
CMV transformation (up to 20 cells per field of view), then this process
was accompanied by extensive fibrosis but uncommonly led to the
formation of a 'honeycomb-like' structure (Fig. 10). The
outcome of CMV infection of the lungs was peribronchial and widespread
interstitial fibrosis with thickening and vast deformation of the
interalveolar septa.

Mycoses

Characteristic gross findings of candidiasis were found in the
pharynx, larynx, and trachea with invasion into principle bronchi, which
included a pseudomembranous form with white, elevated mucosal plaques.
Bronchopulmonary aspergillosis and candidiasis were characterized by the
collection of fungal mycelium in the lumen of small bronchi and invasion
of fungus into the acini. Candida microabscesses were common and they
had a typical polymorphonuclear leucocytic infiltration. Histologically,
Candida organisms could be identified by their size, budding property,
and pseudohyphae.

The pseudohyphae could be distinguished from Aspergillus hyphae by
the lack of branching, the smaller size, and frequent absence of true
septations in the former (Fig. 11). Results were confirmed using the
Romanovskii staining technique, which is used to differentiate between
Candida and Aspergillus. Bronchopulmonary Aspergillosis was
characterized by the collection of mycelia of Aspergillus in the
bronchial lumen with involvement of the bronchial wall and further
invasion of the fungus into the acini (Fig. 12).

[FIGURE 11 OMITTED]

[FIGURE 12 OMITTED]

Combinations of Opportunistic Infections

The pathological processes found in the respiratory tracts are
summarized in Fig. 13.

[FIGURE 13 OMITTED]

One peculiarity of the course of opportunistic pathologies in this
series was simultaneous combined infections of the lungs (28% of
patients). The most frequent was the combination of tuberculosis with
CMV infection (two patients) and Pneumocystis carinii pneumonia with CMV
infection (one patient). There was a wide spectrum of combined
infections of respiratory organs and diverse types of combinations of
two or more infections, such as tuberculosis with CMV, Pneumocystis
carinii pneumonia and purulent bacterial pneumonia, and candidiasis with
Pneumocystis carinii pneumonia in three cases. Histopathology revealed
alternation of some of the various infections, as in focal changes,
localized in various lobes or segments of lungs.

HIV-Associated Neoplasia

About 30% to 40% of patients with HIV infection develop a
malignancy during their lifetime (Spano, Atlan, Breau, & Farge,
2002). Most cancers affecting HIV-positive patients are those
established as AIDS-defining: Kaposi's sarcoma, non-Hodgkin's
lymphoma (NHL), and invasive cervical cancer. Analyses have revealed a
two- to threefold increase in the overall risk of developing these
cancers (Mbulaiteye, Biggar, Goedert, & Engels, 2003). NHL
encompasses several types of lymphoma, including systemic NHL, primary
central nervous system NHL (also referred to as primary brain lymphoma
or cerebral lymphoma), primary effusion lymphoma (PEL), or body
cavity-based lymphoma. We found two cases of high-grade systemic NHL
affecting predominantly the spleen, liver, and bone marrow and these
were the main causes of death.

Women infected with HIV are more likely to be coinfected with human
papilloma virus, possibly because of similar risk profiles and mode of
transmission (Palefsky, Holly, Ralston, & Jay, 1998). We detected
one case of a well-differentiated cervical squamous cell carcinoma that
had invaded the uterus, perforating its wall and leading to peritonitis
causing death of the patient. At autopsy, metastases were detected in
the liver, lung, and spleen.

Conclusion

The main cause of death of these 32 patients with HIV infection was
infectious diseases, in which tuberculosis was the most widespread.
Moreover, it had affected multiple organs and had a progressive course
of disease with a predominance of miliary tuberculosis. We rarely
encountered tuberculous meningitis and peritoneal infection with evident
morphological signs of peritonitis. Destructive processes and
predominantly alterative and exudative reactions in tuberculous
inflammation were typical in all cases. Simultaneous involvement of
organs with many infectious processes was a characteristic feature in
these patients. Infections such as CMV and Pneumocystis carinii
pneumonia were revealed at the autopsy, which had not been treated and
hence had progressed in their disease course. The respiratory tract was
the most affected organ with a prevalence of tuberculosis and pneumonia
of various etiologies. The patients that were studied here were mostly
young men, which is understandable as they are a high-risk group tending
to use unprotected sexual activity and with a high frequency of
intravenous drug use. Moreover, most of the patients had been diagnosed
with HIV in prison.